Project Summary/Abstract Primary central nervous system lymphoma (PCNSL) is an aggressive disease that presents solely in the central nervous system (CNS). Due to its low disease incidence and the poor quality of patient samples, it has very been challenging to establish preclinical disease models of PCNSL. Therefore, a majority of the knowledge we currently have regarding PCNSL biology and pathogenesis is based on sequencing studies in small patient cohorts, particularly gene expression studies and mutation profiling. Based on gene expression studies, most PCNSL is categorized as diffuse large B cell lymphoma (DLBCL), the most common form of Non-Hodgkin lymphoma. Of the three DLBCL subtypes, PCNSL most resembles the activated B cell-like (ABC) subtype. Based on mutation profiling of PCNSL tumors, PCNSL has recurrent mutations in myeloid differentiation primary response gene 88 (MYD88) (70%) and cluster of differentiation 79B (CD79B) (40%). MYD88 is an adaptor protein associated with Toll-like receptor (TLR), while CD79B is a molecule required for B cell receptor (BCR) signaling. MYD88 and CD79B mutations are also found in ABC DLBCL, though not to the same frequencies as in PCNSL. These mutations are associated with increased activation of nuclear factor kappa-light-chain enhancer of activated B cells (NF-?B) in PCNSL. Several targeted therapies have been developed to inhibit this pathway and one example (the first in class) is ibrutinib, a Bruton's Tyrosine Kinase (BTK) inhibitor. BTK is a central signaling node downstream of BCR and TLR. BTK inhibition with ibrutinib has recently been FDA approved for the treatment of several B cell malignancies. Our Phase I/II clinical trial at Memorial Sloan Kettering Cancer Center in relapsed or refractory CNS lymphoma showed that 75% of patients responded to ibrutinib. Though this may appear to simply be a B cell lineage addiction to BTK, we think this is unlikely because some B cell malignancies actually do not respond to ibrutinib. Moreover, of the study patients who responded to ibrutinib, some had mutations in MYD88 and/or CD79B while others did not, indicating that MYD88/CD79B mutation status is insufficient to explain the ibrutinib response. We therefore hypothesize that PCNSL has an overall dependency on BTK that is not due to a B cell lineage addiction. In the proposal, we aim to understand the BTK dependency of PCNSL by assessing the relationship between BTK activation and ibrutinib response in our novel preclinical disease models, determining the effect of the microenvironment on BTK dependency, and exploring mechanisms of ibrutinib resistance and combination therapies to overcome resistance. Our overall goal is to further understand the role of BTK in the PCNSL response to ibrutinib, so that we can take steps to augment the positive results we have seen in patients.